hucMSC treatment ameliorated pulmonary fibrosis via downregulating the circFOXP1–HuR–EZH2/STAT1/FOXK1 autophagic axis

doi:10.21203/rs.3.rs-1494358/v1

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Research Article

hucMSC treatment ameliorated pulmonary fibrosis via downregulating the circFOXP1–HuR–EZH2/STAT1/FOXK1 autophagic axis

https://doi.org/10.21203/rs.3.rs-1494358/v1

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Background

Currently, no effective treatment has been developed for pulmonary fibrosis. This study was performed to determine the effect of human umbilical cord mesenchymal stem cell (hucMSC) treatment on pulmonary fibrosis and investigate the circFOXP1-mediated mechanism of hucMSC treatment on autophagy.

Methods

Pulmonary fibrosis models were established by spraying bleomycin in mice and TGF-β1 treatment of MRC-5 cell. The effect of hucMSC treatment was investigated using morphological and functional experiments. The mechanism of hucMSC treatment in pulmonary fibrosis was explored by gain- and loss-of-function studies and rescue experiments.

Results

Results showed that hucMSC treatment alleviated pulmonary fibrosis. Morphological staining indicated that hucMSC-treated mice had thinner alveolar wall, effectively improved alveolar structure, significantly reduced alveolar inflammation, and decreased collagen deposition than control mice. Fibrotic proteins, including vimentin, α-SMA, collagen I, and collagen III, and the differentiation-related protein S100 calcium binding protein A4 were reduced considerably in the hucMSC-treated group. The mechanistic study revealed that the inhibition of hucMSC treatment on pulmonary fibrogenesis depended on downregulating circFOXP1, in which hucMSC treatment promoted circFOXP1-mediated autophagy process via blocking the nuclear HuR translocation and promoting the HuR degradation, leading to a marked decrease in autophagy negative regulators EZH2, STAT1 and FOXK1.

Conclusion

hucMSC treatment significantly improved pulmonary fibrosis by downregulating the circFOXP1–HuR–EZH2/STAT1/FOXK1 autophagic axis. hucMSCs can act as an effective treatment for pulmonary fibrosis.

pulmonary fibrosis

human umbilical cord mesenchymal stem cells

autophagy

circRNA

HuR

Pulmonary fibrosis is defined as an excessive, pathologic deposition of the extracellular matrix during lung wound healing and is characterized by destroyed lung parenchyma and architecture, lost compliance, and compromised gas exchange [1]. Pulmonary fibrosis is not one disease but is an end-stage state of various diseases. Idiopathic pulmonary fibrosis (IPF) is the most prevalent and morbid of all types. Although the underlying mechanisms of pulmonary fibrosis, such as genetics, epigenetics, and metabolism, have been revealed, treatment options are limited. Mainstay therapies are the oral antifibrotic drugs pirfenidone and nintedanib, which can decrease the collagen deposition, attenuate symptoms, and lower the rate of decline in forced vital capacity. However, these therapies have some bad side effects, such as skin rash, weight loss, nausea, and fatigue, and abnormal liver function [2]. At present, lung transplantation may be the only treatment to increase life expectancy but is challenging due to several concerns, such as risk of mortality and likelihood of survival. Furthermore, other comorbidities that may lead to complications, such as cardiac dysfunction, diabetes, and obesity, are key considerations [3]. The lack of curative treatment demands the aggressive pursuit of improved choices for pulmonary fibrosis. Glass DS et al. proposed that mesenchymal stem cells (MSCs) will be the future treatment for pulmonary fibrosis [4]. The Human Autologous Lung Stem Cell Transplant for IPF (NCT04262167), a randomized trial of intravenous infusion of lung stem cells in patients with IPF, is currently enrolling. However, the knowledge of MSCs towards pulmonary fibrosis is still insufficient.

MSCs are a heterogeneous subset of stromal stem cells that can induce peripheral tolerance and migrate to injured tissues [5]. Human umbilical cord MSCs (hucMSCs) are a promising source of MSCs and attractive autologous or allogenic agents for disease treatment. hucMSC transplantation facilitates locomotor function, promotes tissue repair, and inhibits demyelination in spinal cord injury rats [6]. The intravenous transplantation of hucMSCs is a salvage and priority treatment option for severe COVID-19 [7]. The transplantation of hucMSCs can also treat premature ovarian failure [8]. Hu Y et al. obtained extracellular vesicles (EVs) from the conditioned medium of hucMSCs. Their results in vivo and in vitro illustrated that hucMSC-EVs can serve as a potential agent for the prevention and treatment of osteoporosis [9]. In addition, other studies reported that hucMSCs attenuate pulmonary fibrosis via different signal pathways [10–12]. However, the roles and mechanisms of circRNAs in the process of hucMSCs attenuating pulmonary fibrosis are largely unknown.

circRNAs exhibit cell-/tissue-specific development and stage-specific characteristics in multiple fundamental biological processes physiologically and pathologically, suggesting that circRNAs can be used as potential biomarkers for disease diagnosis and therapy. Recent studies demonstrated correlations between circRNA and autophagy in many diseases. For example, autophagy-associated circCDYL is screened by circRNA deep sequencing and can augment autophagy and promote breast cancer progression [13]. Autophagy-related circACR represses autophagy and myocardial infarction by targeting the Pink1-mediated phosphorylation of FAM65B [14]. However, the relationship between hucMSC treatment and autophagy-associated circRNA in pulmonary fibrosis remains largely illusive.

In our previous study [15], the crosstalk of mRNA, miRNA, lncRNA, and circRNA and their regulatory pattern in pulmonary fibrosis have been elucidated. The circ057 is formed by the circularization of the sequence of 9–10 exons in forkhead box protein 1 (FOXP1) gene and functions as a profibrotic factor. In this study, we name circ057 as circFOXP1 and explore its use as a therapeutic autophagy-related gene (ATG) for hucMSC treatment in pulmonary fibrosis.

hucMSCs isolation and culture

Umbilical cord of full-term newborn was taken aseptically from healthy puerpera with cesarean section, cut off the ligated ends, and washed several times with phosphate-buffered saline (PBS). Each umbilical cord was cut to 1-1.5 cm long, peeled off two umbilical arteries, one umbilical vein and umbilical cord capsule, and further minced into 1 mm³ fragments. The fragments were cultured in 10 mL Dulbecco's modified Eagle's medium containing low glucose (DMEM-LG, Hyclone, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, USA), 100 U/mL penicillin G and 100 U/mL streptomycin sulfate and incubated at 37°C in a humidified atmosphere with 5% CO₂. The medium was replaced per 3 days. Cultured cells at their third passage were tested for the expression of phenotypes such as human leukocyte antigen (HLA)-DR, CD90, CD73, CD44, CD45, CD34 (Milteni Biotec, Germany) and were cultured with osteogenic or adipogenic differentiation medium to establish cells quality control. Primary hucMSCs were cultured to 4-6 generations for the next experiment.

Adipogenic and osteogenic differentiation

The cells were seeded in 24-well plates. When the cells were fused to 90-95%, the medium was replaced by adipogenic or osteogenic differentiation medium. The methods of differentiation were carried out according to MesenCult™ Differentiation Kit (Human). The cells were cultured in 37 °C，5% CO₂ incubator. The medium was replaced per 3 days. After induction of 4 weeks or 2 weeks, oil red O and alizarin red staining were performed and observed under microscope.

Flow cytometry analysis

The cells were collected and centrifuged at 1000 rpm for 5 min. After supernatant was discarded, PBS was added to resuspend precipitate. HLA-DR, CD34, CD45, CD73, CD90 and CD44 antibodies were added, respectively. After incubation at 4℃ for 30 min, 1 mL PBS was added to each sample to wash away the unbound antibodies. Flow cytometry was used to detect it.

Animal model

Eight-week-old C57BL/6 mice (average weight = 20 ± 5 g) were purchased from Jinan Tengli Company. All animal experiments were approved by the Animal Protection and Ethics Committee of Binzhou Medical University. Mice were randomly divided into four groups with 10 mice in each group: sham, bleomycin (BLM) treatment, BLM + hucMSC treatment, and hucMSC treatment groups. Mice were anesthetized by the intraperitoneal injection of 4% chloral hydrate. The Penn–Century MicroSprayer (Penn-CenturyInc.,Wyndmoor, PA, USA) was used to spray 5 mg/kg BLM in the fibrotic model group. About 0.1 mL hucMSCs (10⁶/mL) were injected into mice in the hucMSC treatment group through the tail vein after three days of BLM spraying. hucMSCs were injected in the hucMSC control group, and the sham group was injected with the same amount of normal saline. After 28 days of modeling, the lung tissue of the right lobe of mice was frozen in liquid nitrogen to extract tissue protein and RNA. The lung tissue of the left lobe was perfused and fixed with 4% paraformaldehyde for follow-up experiments.

Hematoxylin-eosin (H&E) and Masson staining

The fixed mice lung tissues were dehydrated, waxed overnight and embedded in paraffin. 4 μM slices were obtained with the Leica (RM2255) slicing machine and stained with H&E and Masson trichrome staining kit (Solarbio, China), respectively. The stained slices were dehydrated with anhydrous ethanol, made transparent with xylene, dropped the neutral gum and sealed with the cover glass. Then lung tissues in each group were observed under light microscope.

Cell model

Human fetal lung fibroblast MRC-5 cell lines were obtained from the American Type Culture Collection and cultured in advanced minimum essential medium supplemented with 10% fetal bovine serum, 1% Gluta-max, 1% NEAA, 1% sodium pyruvate, and 1% 100× penicillin/streptomycin solution at 37 °C and atmosphere containing 5% CO₂. Cells were divided into four groups: normal, TGF-β1 treatment, TGF-β1 + hucMSC treatment, and hucMSC treatment groups. Cells were treated with and without 5 ng/mL TGF-β1 and/or hucMSC supernatant for 48 h except for special instructions.

hucMSCs supernatant preparation: The complete DMEM medium was replaced by serum-free DMEM medium until the fourth passages of hucMSCs reached 70-80% confluence. After cultured for 48 hours, hucMSCs and medium were collected and centrifuged at 1000 rpm for 5 minutes to remove the dead cells and debris. The supernatant of hucMSCs was collected after the medium being filtered by a 0.22-μM syringe filter.

Western blot

Cells or lung tissues were collected and lysed in radioimmunoprecipitation assay (RIPA) buffer and phenylmethylsulfonyl fluoride (PMSF) (RIPA buffer: PMSF=100:1). The protein concentration was measured by bicinchoninic acid protein assay kit（Coolaber，China). After separated in sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was transferred to polyvinylidenefluoride membranes. The protein was sealed 2 hours with 5% skim milk and incubated overnight at 4℃ with anti-collagen Ⅰ, anti-collagen Ⅲ, anti-vimentin, anti-α-SMA, anti-ULK1, anti-ATG5, anti-GABARAP (1:1000, Affinity, China), anti-GAPDH (1:10,000, Affinity, China), anti-P62, anti- LC3, anti-EZH2, anti-FOXK1, anti-STAT1 (1:1000,Abcam,UK), anti-DRAM 2 (1:1000, Bioss,China), anti-HuR (1:2000, Proteintech, China) polyclonal antibodies. 1× Tris Buffered Saline Tween was used to wash the membranes for three times and then incubated 1 hour with secondary antibodies at room temperature. The expression of protein was detected by enhanced chemiluminescence reagent kit (Spark Jade, China). Quantitative analysis of protein bands was performed by Image J software.

Dual-fluorescence HBAD-mcherry-EGFP-LC3 detection

MRC-5 cell samples were seeded in glass cell culture dish. When the cell density reached 60-70%, 500 μL serum-free medium replaced the complete medium and 5 μL HBAD-mcherry-EGFP-LC3 (HANBIO, China) was added. After hatching for 6 h, the medium contained HBAD-mcherry-EGFP-LC3 was removed. Then, the cell samples were treated with/without TGF-β1 and/or hucMSCs supernatant for 36 h. The fluorescence images were detected under a laser scanning confocal microscope (Zeiss LSM880，Germany).

Statistical analysis

Data were expressed as the means ± standard deviation (SD) and analyzed using the SPSS 21.0 statistic software program. One-way analysis of variance (ANOVA) was used to assess overall differences among the groups with post hoc comparisons. P value of < 0.05 was considered statistically significant

Culture and identification of hucMSCs

First, hucMSCs were isolated and cultured. No change in the morphology of umbilical cord was observed at the initial stage. With prolonged culture time, the tissue mass was condensed into a ball, and scattered transparent dots were observed around the tissue block. Transparent dots gradually became long, and the number gradually increased. On the ninth day after the tissue block adhered to the wall, spindle cells gathered around the tissue block. On the 12th day, the cells around the tissue block fused into pieces (Fig. 1a). Then, osteogenic and adipogenic differentiation, and flow cytometry were used to identify hucMSCs. The third generation of hucMSCs was used to induce osteogenic and adipogenic differentiation. After four weeks of adipogenic induction, fat droplets of verified size were observed using phase-contrast microscopy with positive oil red O staining (Fig. 1b). After two weeks of osteogenic induction, calcium nodus alizarin red staining reaction was positive with the osteogenic inducer (Fig. 1c). The flow cytometry of cell surface markers demonstrated that the cells expressed CD73, CD90, and CD44 and not CD34, CD45, and human leukocyte antigen DR (HLA-DR, Fig. 1d). The above experimental results indicated that cells had the main features of MSCs and confirmed that hucMSCs had been successfully isolated and cultured in vitro, providing hucMSCs for pulmonary fibrosis treatment.

hucMSC treatment ameliorated pulmonary fibrosis via downregulating circFOXP1 in vivo and in vitro

The preventive effect of hucMSC treatment on pulmonary fibrosis was examined in bleomycin (BLM)-treated mice and in TGF-β1-treated human fetal lung fibroblast (MRC-5 cell). H&E and Masson staining results exhibited thickened alveolar wall, widened alveolar septum, large number of fibroblasts, and increased collagen deposition in the BLM-treated group. After the tail vein injection of hucMSCs, mouse lungs had thinner alveolar wall, effectively improved alveolar structure, significantly reduced alveolar inflammation, and decreased collagen deposition compared with BLM-treated mice (Figs. 2a–b). Western blot results illustrated that hucMSCs induced a rapid decrease in fibrotic proteins, including vimentin, α-SMA, collagen I, and collagen III. The differentiation-related protein S100 calcium binding protein A4 (S100A4) was also decreased by hucMSCs (Fig. 2c). The above results showed that hucMSC treatment ameliorated pulmonary fibrosis in vivo.

Next, the TGF-β1-treated MRC-5 cell model was established to further examine the preventive effect and mechanism of hucMSC treatment on pulmonary fibrosis. CCK-8 results indicated that hucMSC treatment inhibited the viability of TGF-β1-treated cells (Fig. 3a). Cell growth curves verified that hucMSC treatment prevented TGF-β1-treated cell proliferation by using a real-time cell analyzer (Fig. 3b). The cell scratch assay showed that the migration was promoted by TGF-β1 treatment and repressed by hucMSC treatment (Fig. 3c). Immunofluorescent images and Western blot exhibited that the hucMSC-treated group had substantially reduced α-SMA, vimentin, collagen Ⅰ, collagen III, and S100A4 compared with the TGF-β1-treated group (Figs. 3d–e). The above results showed that hucMSC treatment ameliorated pulmonary fibrosis in vitro.

We further investigated the inhibitory effect of hucMSC treatment on pulmonary fibrosis through circFOXP1. First, circFOXP1 expression was detected under hucMSC action in vivo and in vitro. qRT-PCR results illustrated that hucMSC treatment inhibited circFOXP1 expression (Fig. 4a). The nuclear–cytoplasmic separation showed that circFOXP1 was located in the cytoplasm with or without TGF-β1 and/or hucMSC treatment (Fig. 4b). Gain- and loss-of-function studies revealed that circFOXP1 knockdown (si-circFOXP1) reduced the expression levels of vimentin, α-SMA, and collagens Ⅰ and III. The circFOXP1 overexpression enhanced the expression of these fibrotic proteins (Fig. 4c). The rescue experiment showed that the circFOXP1 overexpression reversed the downward trend of these fibrotic proteins caused by hucMSC treatment (Fig. 4d), which implied that the inhibitory effect of hucMSC treatment on fibrosis depended on circFOXP1 in vitro.

Finally, the reduction of pulmonary fibrosis by hucMSC treatment via circFOXP1 was explored in mice. We synthesized the circFOXP1 overexpression packaged into adenovirus vectors and sprayed to the lungs of mice. The rescue experiment of the microcomputed tomography (CT) imaging system for small animals demonstrated that BLM caused evident fibrosis in both lower lungs, and hucMSCs alleviated the degree of fibrosis, and circFOXP1 upregulation reversed the inhibitory effect of hucMSCs on fibrosis (Fig. 5a). The rescue experiments of H&E and Masson staining further proved that thickened alveolar walls and increased collagen deposition in the lung tissue of BLM-treated mice. hucMSCs alleviated morphological abnormalities, and circFOXP1 upregulation reversed the inhibitory effect of hucMSCs (Fig. 5b). The above findings confirmed that hucMSC treatment alleviated pulmonary fibrosis by downregulating circFOXP1 in vivo.

circFOXP1-mediated inhibition of hucMSC treatment on pulmonary fibrosis by promoting human antigen R (HuR)-mediated autophagy process

In our previous study [16], we reported that autophagy is repressed in pulmonary fibrosis. Thus, we investigated the circFOXP1-mediated mechanism of hucMSC treatment on autophagy during pulmonary fibrogenesis. Microtubule-associated protein 1 light chain 3 (LC3) is commonly used to label autophagosome. Thus, the tandem dual-fluorescence HBAD–mcherry–EGFP–LC3 was performed to confirm the regulation of hucMSC treatment on autophagy via circFOXP1. Images depicted that the normal group emitted red fluorescence, the TGF-β1 group emitted green fluorescence, and the hucMSC-treated group emitted yellow fluorescence (Fig. 6a), which implied that hucMSC treatment enhanced the autophagy process. The rescue experiment of dual-fluorescence HBAD–mcherry–EGFP–LC3 showed that upregulated circFOXP1 caused green fluorescence enhancement and reversed the enhanced effect of hucMSC treatment on autophagy (Fig. 6b). Findings proved the promoting effect of hucMSC treatment on autophagy via downregulating circFOXP1.

HuR can negatively regulate autophagy in pulmonary fibrosis. Thus, the effect of hucMSC treatment on HuR was assessed. Western blot results discovered that hucMSC treatment substantially reduced the HuR expression level in vivo and in vitro (Fig. 7a). The small interfering RNA of circFOXP1 also reduced HuR, and circFOXP1 overexpression promoted HuR (Fig. 7b). Nuclear–cytoplasmic separation results showed that HuR was located in the nucleus of normal cells and transferred to the cytoplasm under TGF-β1 treatment, whereas the hucMSC treatment blocked the transfer process (Fig. 7C). The rescue experiment of nuclear–cytoplasmic separation demonstrated that circFOXP1 upregulation reversed the inhibitory effect of hucMSC treatment on HuR nucleocytoplasmic shuttling (Fig. 7d), which suggested the inhibitory effect of hucMSC treatment on nuclear HuR translocation via inhibiting circFOXP1. Immunofluorescence images further exhibited that HuR was located in the nucleus of normal cells and transferred to the cytoplasm after being treated with TGF-β1, whereas hucMSC treatment inhibited the transfer process (Fig. 7e). The rescue experiment of immunofluorescence images depicted that circFOXP1 upregulation reversed the inhibitory effect of hucMSC treatment on nuclear HuR nucleocytoplasmic shuttling (Fig. 7f). Findings further proved the effect of hucMSC treatment on nuclear HuR translocation through circFOXP1. The stability of HuR was performed using cycloheximide experiment, and results discovered that TGF-β1 promoted HuR stability and that HuR stability was weakened by hucMSC treatment. The rescue experiment elucidated that the weakening trend caused by hucMSC treatment was reversed by overexpressed circFOXP1, which implied that the effect of hucMSC treatment on HuR stability also depended on circFOXP1 (Fig. 7g). The above findings indicated that hucMSC treatment blocked nuclear HuR translocation from nucleus to cytoplasm. Besides, hucMSC treatment promoted HuR degradation via downregulating circFOXP1.

hucMSC treatment promoted autophagy by targeting enhancer of zeste homolog 2 (EZH2), signal transducers and activators of transcription 1 (STAT1), and forkhead box K1 (FOXK1)

HuR can repress autophagy by controlling the target genes associated with autophagy, such as EZH2, STAT1, and FOXK1 [16]. Then, the effects of hucMSC treatment on autophagy through the target genes EZH2, STAT1, and FOXK1 were further explored. Western blot results demonstrated that hucMSC treatment reduced the expression levels of EZH2, STAT1, and FOXK1 in vivo and in vitro. Given that autophagic flux activated autophagy, autophagic flux marker proteins, such as LC3-II/LC3-I and P62, were also detected by Western blot. P62 and LC3-II/LC3-I levels decreased in the normal group. P62, LC3-II, and LC3-I levels increased in the TGF-β1/BLM-treated group compared with those in the normal group. P62, LC3-II, and LC3-I levels in the hucMSC-treated group decreased compared with those in TGF-β1/BLM-treated group (Fig. 8a). Gain- and loss-of-function studies revealed that si-circFOXP1 had the same effect as hucMSC treatment, whereas circFOXP1 overexpression had the opposite effect of hucMSC treatment (Fig. 8b). This finding implied that hucMSC treatment and si-circFOXP1 enhanced autophagic flux via EZH2, STAT1, and FOXK1. The rescue experiment of circFOXP1 overexpression further elucidated that the enhancement effect of hucMSC treatment on the target genes and autophagic flux depended on circFOXP1 (Fig. 8c).

The above findings demonstrated that autophagic flux was blocked, but the autophagosome accumulation was significantly increased in pulmonary fibrosis (Figure 6). Thus, we inferred that autophagy was blocked in the later stage and hucMSC treatment promoted autophagy process in pulmonary fibrosis. Next, the effects of hucMSC treatment on the autophagy process were further analyzed by the expression of ATGs. Unc-51-like kinase 1 (ULK1) and ATG5 contributed to the early stage of autophagy, and DRAM2 and GABARAP helped in the autophagy via the autophagosome–lysosome fusion in the later stage. Western blot analysis revealed that ULK1 and ATG5 were increased by TGF-β1 but decreased by hucMSC treatment in vivo and in vitro. DRAM2 and GABARAP levels were decreased by TGF-β1 but increased by hucMSC treatment in vivo and in vitro (Fig. 8d). This finding confirmed that hucMSC treatment promoted the autophagy process. Western blot analysis revealed that the effect of si-circFOXP1 was consistent with hucMSC treatment. Thus, si-circFOXP1 reduced EZH2, STAT1, FOXK1, P62, LC3-II, LC3-I, ULK1, and ATG5 expression levels and enhanced DRAM2 and GABARAP expression levels. The effect of circFOXP1 overexpression was opposite to that of si-circFOXP1 (Fig. 8e). The rescue experiment of Western blot indicated that circFOXP1 overexpression reversed the effect of hucMSC treatment on ULK1, ATG5, DRAM2, and GABARAP levels, which implied the effect of hucMSC treatment on ATGs via circFOXP1 (Fig. 8F).

Currently, no effective therapeutic drug in the Official Clinical Practice Guideline has been recommended for pulmonary fibrosis [17–19]. Clearly, the need for accelerated research into IPF treatment is unmet. hucMSCs have emerged as important stem cell therapy for disease due to their high differentiation potential, painless and easy collection procedure, minimal stringent requirements for HLA matching, and no ethical dilemma [20]. Zhang C et al. described a case of a 56-year-old man with IPF who underwent hucMSC intravenous infusion and was followed up for 12 months [21]. No adverse event is observed at 12 months post-infusion, and lung function, 6-minute walking distance, and CT fibrosis score have increased from baseline. This case report provides useful clinical insight for the future efficacy trial of stem cell therapy in pulmonary fibrosis. However, the therapeutic mechanism of hucMSC treatment in pulmonary fibrosis remains poorly understood, preventing its clinical application. In the present study, cultured hucMSCs are injected into the pulmonary fibrosis mice via the tail vein to investigate their role. Results illustrated that hucMSC treatment improves the alveolar structure, reduces the deposition of collagen fibers, and decreases the differentiation-related protein. Mechanistic studies in vivo and in vitro revealed that hucMSC treatment ameliorates pulmonary fibrosis via downregulating the circFOXP1–HuR–EZH2/STAT1/FOXK1 autophagic axis.

The autophagy process clears damaged organelles, intracellular abnormal proteins, or invading microorganisms. Dysfunctional autophagy causes the pathological accumulation of the cargo, which leads to human diseases, such as infectious diseases, cancer, and neurodegenerative diseases [22]. Hence, drugs that potentially modulate autophagy are increasingly being used in clinical trials, and screening for new drugs that can modulate autophagy for therapeutic purposes is being carried out [23]. The role of autophagy on pulmonary fibrosis remains largely unknown. Most studies indicated that the activation of autophagy helps in blocking pulmonary fibrosis [24,25]. Zhang et al. explored that lncIAPF accelerates pulmonary fibrogenesis by blocking autophagosome fusion with lysosome and/or inhibiting the degradation of autophagosomes and showed that the knockdown of lncIAPF can enhance autophagy to attenuate pulmonary fibrosis [16]. However, Tsubouchi K et al. elucidated that Azithromycin alleviates lung fibrosis development by inhibiting autophagy and enhancing the unfolded protein response [26]. To date, the roles of autophagy in the process of hucMSC treatment attenuating pulmonary fibrosis and the mechanisms of hucMSC treatment regulating autophagy are largely unknown. The present study proved that hucMSC treatment activates the autophagy process to ameliorate pulmonary fibrosis via downregulating the circFOXP1–HuR–EZH2/STAT1/FOXK1 autophagic axis.

HuR is a potential therapeutic target candidate-related autophagy for disease therapy [27–29]. However, inhibiting HuR may cause many side effects because of the central functions of HuR, such as controlling mRNA stability and the expression of various cytokines, proto-oncogenes, and growth factors. circRNA is usually expressed at lower levels than mRNA or protein. Therefore, circRNA-targeted treatment has more advantages than directly targeting mRNA or protein. For example, astilbin attenuates pulmonary fibrosis via circRNA-662 and 949 sponging the miR‐29b targeting the Gli2 and STAT3 signal pathway [30]. circANKRD42 mediates the crosstalk of mechanical stiffness and biochemical signal in lung fibrosis, and the knockdown of circANKRD42 can attenuate pulmonary fibrosis [31]. Now, research articles about the relationship of hucMSC treatment and circRNA are rare. In several articles, only the regulation of circRNA on hucMSC differentiation, not hucMSC treatment, is discussed [32–35]. The role and mechanism of circRNA in hucMSC treatment have not been reported. The present study illustrated that hucMSC treatment ameliorates pulmonary fibrosis via blocking nuclear HuR translocation from nucleus to cytoplasm and promoting HuR degradation depending on circFOXP1. EZH2, STAT1, and FOXK1 are the target genes of HuR, as proven by HuR RNA-binding protein immunoprecipitation (RIP) sequencing and RIP-PCR [16], and are autophagy negative regulators. This study further elucidated that hucMSC treatment enhances the autophagy process through inhibiting the HuR level, leading to a marked decrease in EZH2/STAT1/FOXK1 expression via circFOXP1.

Our findings showed that hucMSC treatment can alleviate pulmonary fibrosis, which is partly associated with the enhancement of autophagy through downregulating the HuR–EZH2/STAT1/FOXK1 autophagic signal pathway depending on circFOXP1. We hope this study can provide new therapeutic targets and strategies for patients with IPF.

MSCs

mesenchymal stem cells

hucMSCs

human umbilical cord mesenchymal stem cells

S100A4

S100 calcium binding protein A4

ECM

extracellular matrix

IPF

idiopathic pulmonary fibrosis

FVC

forced vital capacity

HALT-IPF

human autologous lung stem cell transplant for IPF

EVs

extracellular vesicles

FOXP1

forkhead box protein 1

HLA-DR

human leukocyte antigen DR

LC3

microtubule-associated protein 1 light chain 3

HuR

Human antigen R

EZH2

the enhancer of zeste homolog 2

STAT1

signal transducers and activators of transcription 1

FOXK1

forkhead box K1

ATGs

the autophagy-related genes

ULK1

Unc-51-like kinase 1

computed tomography

UPR

the unfolded protein response

RIP-seq

RNA binding protein immunoprecipitation sequencing

PBS

phosphate-buffered saline

FBS

fetal bovine serum

BLM

bleomycin

H&E

Hematoxylin-eosin

MRC-5

Human fetal lung fibroblast

MEM

minimum essential medium

RIPA

radioimmunoprecipitation assay

PMSF

phenylmethylsulfonyl fluoride

Acknowledgements

All authors are acknowledged for their contribution to the study.

Authors’ contributions

LME, LHB and LCJ contributed to the conception and design of the work. LRQ, ZHT and ZJJ contributed to perform the experiment and data analysis. SXD, LB, JYX and ZNL were contributors in the database search and preparing the manuscript. The authors read and approved the ending version of the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (82170085, 81970064, 81870001, 81670064, and 31670365), the Natural Science Foundation of Shandong Province (ZR2020MH009, ZR2020MH010, ZR2019PH101, 2019WS325), and the regenerative medicine research program of Xu Rongxiang (50012305179).

Availability of data and materials

All data generated and/or analyzed during this study are included in this published article.

Ethics approval and consent to participate

Animals were treated in accordance with the Basel Declaration in the context of phase experimental animals. The use of animals was approved by the Ethics Committee of Binzhou Medical University. We have obtained the participants’ informed consent to donate the tissue for research use. The use of the human tissue was approved by the Ethics Committee of the Binzhou Medical University Hospital.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests

Author details

1 Department of Clinical Nursing, Binzhou Medical University Hospital, Binzhou Medical University, Binzhou 256603, China.

2 Department of Respiratory and Critical Care Medicine, Binzhou Medical University Hospital, Binzhou Medical University, Binzhou 256603, China.

3 Medical Research Center, Binzhou Medical University, Yantai 264003, China.

4 Department of Cellular and Genetic Medicine, School of Pharmaceutical Sciences, Binzhou Medical University, Yantai 264003, China.

Moss BJ, Ryter SW, Rosas IO. Pathogenic Mechanisms Underlying Idiopathic Pulmonary Fibrosis. Annu Rev Pathol. 2022;17:515–546.
Vancheri C, Kreuter M, Richeldi L, Ryerson CJ, Valeyre D, Grutters JC, et al. Nintedanib with Add-on Pirfenidone in Idiopathic Pulmonary Fibrosis. Results of the INJOURNEY Trial. Am J Respir Crit Care Med. 2018;197(3):356–363.
Young KA, Dilling DF. The Future of Lung Transplantation. Chest. 2019;155(3):465–473.
Glass DS, Grossfeld D, Renna HA, Agarwala P, Spiegler P, DeLeon J, et al. Idiopathic pulmonary fibrosis: Current and future treatment. Clin Respir J. 2022;16(2):84–96.
Uccelli A, Moretta L, Pistoia V. Mesenchymal stem cells in health and disease. Nat Rev Immunol. 2008;8(9):726–736.
Liao Z, Yang X, Wang W, Deng W, Zhang Y, Song A, et al. hucMSCs transplantation promotes locomotor function recovery, reduces apoptosis and inhibits demyelination after SCI in rats. Neuropeptides. 2021;86:102125.
Shu L, Niu C, Li R, Huang T, Wang Y, Huang M, et al. Treatment of severe COVID-19 with human umbilical cord mesenchymal stem cells. Stem Cell Res Ther. 2020;11(1):361.
Shareghi-Oskoue O, Aghebati-Maleki L, Yousefi M. Transplantation of human umbilical cord mesenchymal stem cells to treat premature ovarian failure. Stem Cell Res Ther. 2021;12(1):454.
Hu Y, Zhang Y, Ni CY, Chen CY, Rao SS, Yin H, et al. Human umbilical cord mesenchymal stromal cells-derived extracellular vesicles exert potent bone protective effects by CLEC11A-mediated regulation of bone metabolism. Theranostics. 2020;10(5):2293–2308.
Tang Z, Gao J, Wu J, Zeng G, Liao Y, Song Z, et al. Human umbilical cord mesenchymal stromal cells attenuate pulmonary fibrosis via regulatory T cell through interaction with macrophage. Stem Cell Res Ther. 2021;12(1):397.
Zhang H, Wang H, Xia Y, Qi N. Dual Effects of Hypoxia-Inducible Factors-1 Alpha in Bleomycin-Induced Pulmonary Fibrosis Treated by Human Umbilical Cord Mesenchymal Stem Cells. Stem Cells Int. 2021;2021:6658855.
Liu J, Peng D, You J, Zhou O, Qiu H, Hao C, et al. Type 2 Alveolar Epithelial Cells Differentiated from Human Umbilical Cord Mesenchymal Stem Cells Alleviate Mouse Pulmonary Fibrosis Through β-Catenin-Regulated Cell Apoptosis. Stem Cells Dev. 2021;30(13):660–670.
Liang G, Ling Y, Mehrpour M, Saw PE, Liu Z, Tan W, et al. Autophagy-associated circRNA circCDYL augments autophagy and promotes breast cancer progression. Mol Cancer. 2020;19(1):65.
Zhou LY, Zhai M, Huang Y, Xu S, An T, Wang YH, et al. The circular RNA ACR attenuates myocardial ischemia/reperfusion injury by suppressing autophagy via modulation of the Pink1/ FAM65B pathway. Cell Death Differ. 2019;26(7):1299–1315.
Li C, Wang Z, Zhang J, Zhao X, Xu P, Liu X, et al. Crosstalk of mRNA, miRNA, lncRNA, and circRNA and Their Regulatory Pattern in Pulmonary Fibrosis. Mol Ther Nucleic Acids. 2019;18:204–218.
Zhang J, Wang H, Chen H, Li H, Xu P, Liu B, et al. ATF3-activated accelerating effect of LINC00941/lncIAPF on fibroblast-to-myofibroblast differentiation by blocking autophagy depending on ELAVL1/HuR in pulmonary fibrosis. Autophagy. 2022; DOI:10.1080/15548627.2022.2046448.
Raghu G, Remy-Jardin M, Myers JL, Richeldi L, Ryerson CJ, Lederer DJ, et al. Diagnosis of Idiopathic Pulmonary Fibrosis. An Official ATS/ERS/JRS/ALAT Clinical Practice Guideline. Am J Respir Crit Care Med. 2018;198(5):e44-e68.
Raghu G, Rochwerg B, Zhang Y, Garcia CA, Azuma A, Behr J, et al. An Official ATS/ERS/JRS/ALAT Clinical Practice Guideline: Treatment of Idiopathic Pulmonary Fibrosis. An Update of the 2011 Clinical Practice Guideline. Am J Respir Crit Care Med. 2015;192(2):e3-e19.
Raghu G, Collard HR, Egan JJ, Martinez FJ, Behr J, Brown KK, et al. An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management. Am J Respir Crit Care Med. 2011;183(6):788–824.
Ding DC, Chang YH, Shyu WC, Lin SZ. Human umbilical cord mesenchymal stem cells: a new era for stem cell therapy. Cell Transplant. 2015;24(3):339–347.
Zhang C, Yin X, Zhang J, Ao Q, Gu Y, Liu Y. Clinical observation of umbilical cord mesenchymal stem cell treatment of severe idiopathic pulmonary fibrosis: A case report. Exp Ther Med. 2017;13(5):1922–1926.
Kocak M, Ezazi Erdi S, Jorba G, Maestro I, Farrés J, Kirkin V, et al. Targeting autophagy in disease: established and new strategies. Autophagy. 2021;1–23.
Klionsky DJ, Abdel-Aziz AK, Abdelfatah S, Abdellatif M, Abdoli A, Abel S, et al. Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1. Autophagy. 2021;17(1):1-382.
Wang K, Zhang T, Lei Y, Li X, Jiang J, Lan J, et al. Identification of ANXA2 (annexin A2) as a specific bleomycin target to induce pulmonary fibrosis by impeding TFEB-mediated autophagic flux. Autophagy. 2018;14(2):269–282.
Cabrera S, Maciel M, Herrera I, Nava T, Vergara F, Gaxiola M, et al. Essential role for the ATG4B protease and autophagy in bleomycin-induced pulmonary fibrosis. Autophagy. 2015;11(4):670–684.
Tsubouchi K, Araya J, Minagawa S, Hara H, Ichikawa A, Saito N, et al. Azithromycin attenuates myofibroblast differentiation and lung fibrosis development through proteasomal degradation of NOX4. Autophagy. 2017;13(8):1420–1434.
Assoni G, La Pietra V, Digilio R, Ciani C, Licata NV, Micaelli M, et al. HuR-targeted agents: An insight into medicinal chemistry, biophysical, computational studies and pharmacological effects on cancer models. Adv Drug Deliv Rev. 2022;181:114088.
Liang D, Lin WJ, Ren M, Qiu J, Yang C, Wang X, et al. m6A reader YTHDC1 modulates autophagy by targeting SQSTM1 in diabetic skin. Autophagy. 2021;1–20.
Zhang Z, Yao Z, Wang L, Ding H, Shao J, Chen A, et al. Activation of ferritinophagy is required for the RNA-binding protein ELAVL1/HuR to regulate ferroptosis in hepatic stellate cells. Autophagy. 2018;14(12):2083–2103.
Lu G, Zhang J, Liu X, Liu W, Cao G, Lv C, et al. Regulatory network of two circRNAs and an miRNA with their targeted genes under astilbin treatment in pulmonary fibrosis. J Cell Mol Med. 2019;23(10):6720–6729.
Xu P, Zhang J, Wang M, Liu B, Li R, Li H, et al. hnRNPL-activated circANKRD42 back-splicing and circANKRD42-mediated crosstalk of mechanical stiffness and biochemical signal in lung fibrosis. Mol Ther. 2022;S1525-0016(22)00159-9.
Lin Z, Tang X, Wan J, Zhang X, Liu C, Liu T. Functions and mechanisms of circular RNAs in regulating stem cell differentiation. RNA Biol. 2021;18(12):2136–2149.
Su C, Zheng X, He Y, Long L, Chen W. Transcriptomic profiling and functional prediction reveal aberrant expression of circular RNAs during osteogenic differentiation in human umbilical cord mesenchymal stromal cells. Sci Rep. 2021;11(1):19881.
Ruan ZB, Chen GC, Zhang R, Zhu L. Circular RNA expression profiles during the differentiation of human umbilical cord-derived mesenchymal stem cells into cardiomyocyte-like cells. J Cell Physiol. 2019;10.1002/jcp.28310.
Yang L, Bin Z, Hui S, Rong L, You B, Wu P, et al. The Role of CDR1as in Proliferation and Differentiation of Human Umbilical Cord-Derived Mesenchymal Stem Cells. Stem Cells Int. 2019;2019:2316834.

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hucMSC treatment ameliorated pulmonary fibrosis via downregulating the circFOXP1–HuR–EZH2/STAT1/FOXK1 autophagic axis

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